Metabolic engineering of bio-catalytic cells with novel pathways require expression of one or more heterologous proteins. The introduction of new proteins for cellular synthesis increases the demand for cellular resources which can be detrimental to bio-catalytic cells. Constitutive expression of the novel pathway proteins can impede cell growth rates, which in turn reduces productivity. In addition, differing expression levels and activities of pathway enzymes can result in bottlenecks, which indicate wastage of cellular resources. Strategies for optimization of engineered pathways include static controls such as tuning of promoter strength, ribosome binding sites or copy number of the vector to enable balanced pathway reaction flux and remove bottlenecks, resulting in higher product yield.
Advanced optimization strategies involve dynamic controls where protein expression can be triggered on-demand. Dynamic control using inducible promoters enable the separation of growth phase from production phase, allowing cells to accumulate biomass before channeling resources for formation of desired chemicals. Furthermore, dynamic control via the sensing of key intermediates to regulate protein expression levels empowers the cell to, make real-time adjustments to its metabolic flux when host or environmental conditions change. This facilitates efficient consumption of cellular resources, with optimal protein expression levels at all times. The combination of dynamic control strategies by employing both inducible promoters and sensing-regulation can assist the engineering of bio-catalytic cells with robust cell growth and enhanced pathway productivity.
Concerns regarding energy security, petroleum supply and environmental protection have encouraged the development of advanced biofuels. Fatty acid derivatives, including fatty alcohols, fatty acid esters, alkenes and alkanes, are advanced biofuels as they have properties similar to fossil fuels and can be directly used in existing transportation infrastructure. One approach that has great potential for optimizing fatty acid derivatives producing yeast strains would involve dynamic controls. Coupling of the fatty acid sensor-regulator with an inducible promoter will enable AND-gate dynamic control, where the inducible promoter allows the production phase to be triggered when desired, and the sensor-regulator facilitates real-time metabolic flux corrections. In yeast, the galactose promoters which exhibit tight control and 1000 fold induction ratios are commonly employed as inducible promoters. However, for economically sustainable industrial production of fatty acid derived biofuels, the use of an affordable carbon source, such as lignocellulosic biomass is crucial. Since lignocellulose contains glucose, the galactose promoters are catabolite repressed, rendering them unsuitable for constructing the AND-gate dynamic controller. Hence, fatty acid sensor-regulation functionality needs to be introduced to inducible systems that function in glucose medium.